Dimerization of cGMP-dependent Protein Kinase Iβ Is Mediated by an Extensive Amino-terminal Leucine Zipper Motif, and Dimerization Modulates Enzyme Function

During nitric oxide signaling, type Iα cGMP-dependent protein kinase (PKGIα) activates myosin light chain (MLC) phosphatase through an interaction with the 130-kDa myosin targeting subunit (MYPT1), leading to dephosphorylation of 20-kDa MLC and vasodilatation. It has been suggested that the MYPT1-PKGIα interaction is mediated by the COOH-terminal leucine zipper (LZ) of MYPT1 and the NH2-terminal LZ of PKGIα (HK Surks and ME Mendelsohn. Cell Signal 15: 937–944, 2003; HK Surks et al. Science 286: 1583–1587, 1999), but we previously showed that PKGIα interacts with LZ-positive (LZ+) and LZ-negative (LZ−) MYPT1 isoforms ( 13 ). Interestingly, PKGIα is known to preferentially bind to RR and RK motifs (WR Dostmann et al. Proc Natl Acad Sci USA 97: 14772–14777, 2000), and there is an RK motif within the aa 888–928 sequence of MYPT1 in LZ+ and LZ− isoforms. Thus, to localize the domain of MYPT1 important for the MYPT1-PKGIα interaction, we designed four MYPT1 fragments that contained both the aa 888–928 sequence and the downstream LZ domain (MYPT1FL), lacked both the aa 888–928 sequence and the LZ domain (MYPT1TR), lacked only the aa 888–928 sequence (MYPT1SO), or lacked only the LZ domain (MYPT1TR2). Using coimmunoprecipitation, we found that only the fragments containing the aa 888–928 sequence (MYPT1FL and MYPT1TR2) were able to form a complex with PKGIα in avian smooth muscle tissue lysates. Furthermore, mutations of the RK motif at aa 916–917 (R916K917) to AA decreased binding of MYPT1 to PKGIα in chicken gizzard lysates; these mutations had no effect on binding in chicken aorta lysates. However, mutation of R916K917 to E916E917 eliminated binding, suggesting that one factor important for the PKGIα-MYPT1 interaction is the charge at aa 916–917. These results suggest that, during cGMP-mediated signaling, aa 888–928 of MYPT1 mediate the PKGIα-MYPT1 interaction.

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Dimerization of cGMP-dependent protein kinase 1α and the myosin-binding subunit of myosin phosphatase: role of leucine zipper domains

Cellular Signalling ◽

10.1016/s0898-6568(03)00057-3 ◽

2003 ◽

Vol 15 (10) ◽

pp. 937-944 ◽

Cited By ~ 42

Author(s):

Howard K Surks ◽

Michael E Mendelsohn

Keyword(s):

Protein Kinase ◽

Leucine Zipper ◽

Myosin Phosphatase ◽

Dependent Protein Kinase ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein ◽

Myosin Binding ◽

Binding Subunit

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Abstract 1068: Genetic Evidence for a Central Role of cGMP-Dependent Protein Kinase I Alpha in Regulating Cardiac Hypertrophy In Vivo

Circulation ◽

10.1161/circ.116.suppl_16.ii_213-d ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Robert M Blanton ◽

Mark Aronovitz ◽

Alexandra Dabreo ◽

Richard H Karas ◽

Michael E Mendelsohn

Keyword(s):

Protein Kinase ◽

Cardiac Hypertrophy ◽

Leucine Zipper ◽

Early Mortality ◽

Mutant Form ◽

Dependent Protein Kinase ◽

Cgmp Dependent Protein Kinase ◽

Lv Mass ◽

Dependent Protein ◽

Tibia Length

The nitric oxide-cGMP-dependent protein kinase pathway is a central regulator of cardiovascular physiology. Protein Kinase G I (PKGI), a principle mediator of this pathway, has been implicated as a negative regulator of cardiac hypertrophy, but the specific mechanisms involved are unknown. To test the hypothesis that PKGIα regulates cardiac hypertrophy, we characterized the cardiac phenotype of mice homozygous for a mutant form of PKGIα in which critical amino acids in the N-terminal leucine zipper (LZ) motif have been substituted to disrupt PKGIα LZ binding to specific downstream effector proteins. In the unstressed state, male PKG Iα leucine zipper mutant ( LZM) mice develop progressive LV hypertrophy compared with wild type (WT) littermates with LV mass/tibia length 12.3% greater at 30 weeks of age (p=0.05), and 27% greater at 60 weeks (p=0.001). Compared with WT littermates, the hearts of 30 week old PKGIα mutants are hypercontractile with decreased end systolic diameter (p=0.02) and increased fractional shortening on echocardiography (p=0.03). Invasive hemodynamics demonstrate that LZM mice also have increased LV systolic pressure (p=0.04), developed pressure (p=0.05), and LV dP/dt max (p=0.13). To evaluate the response to hemodynamic stress, cardiac hypertrophy was induced by transaortic constriction (TAC) in male WT and LZM mice. TAC resulted in early mortality in the LZM mice (60%) compared to the WT (19%) mice at 21 days post procedure (p=0.008), with evidence of accelerated LV hypertrophy in the mice that died early (LV mass/tibia length 9.8 mg/mm in the early LZM deaths vs 7.2 mg/mm in WT survivors, p<0.001). Additionally, the LZM early deaths had evidence of congestive heart failure with increased RV mass (RV mass/ tibia length 1.8 mg/mm in LZM early deaths vs 1.2 mg/mm in WT survivors, p<0.05), and increased lung mass (23.1 mg/mm LZM early deaths vs 12.5 mg/mm in WT survivors, p<0.005). These findings support that the N-terminal LZ domain of PKGIα is required for suppression of cardiac hypertrophy. The early mortality following TAC in the LZM mice also suggests a critical role for PKGIα in attenuating pathologic cardiac remodeling, identifying PKGIα as an attractive candidate drug target for prevention of cardiac hypertrophy and failure.

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A substitution in cGMP-dependent protein kinase 1 associated with aortic disease induces an active conformation in the absence of cGMP

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010984 ◽

2020 ◽

Vol 295 (30) ◽

pp. 10394-10405

Author(s):

Matthew H. Chan ◽

Sahar Aminzai ◽

Tingfei Hu ◽

Amatya Taran ◽

Sheng Li ◽

...

Keyword(s):

Protein Kinase ◽

Leucine Zipper ◽

Aortic Aneurysms ◽

Cell Phenotype ◽

Active Conformation ◽

Dependent Protein Kinase ◽

Basal Activity ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein

Type 1 cGMP-dependent protein kinases (PKGs) play important roles in human cardiovascular physiology, regulating vascular tone and smooth-muscle cell phenotype. A mutation in the human PRKG1 gene encoding cGMP-dependent protein kinase 1 (PKG1) leads to thoracic aortic aneurysms and dissections. The mutation causes an arginine-to-glutamine (RQ) substitution within the first cGMP-binding pocket in PKG1. This substitution disrupts cGMP binding to the pocket, but it also unexpectedly causes PKG1 to have high activity in the absence of cGMP via an unknown mechanism. Here, we identified the molecular mechanism whereby the RQ mutation increases basal kinase activity in the human PKG1α and PKG1β isoforms. Although we found that the RQ substitution (R177Q in PKG1α and R192Q in PKG1β) increases PKG1α and PKG1β autophosphorylation in vitro, we did not detect increased autophosphorylation of the PKG1α or PKG1β RQ variant isolated from transiently transfected 293T cells, indicating that increased basal activity of the RQ variants in cells was not driven by PKG1 autophosphorylation. Replacement of Arg-177 in PKG1α with alanine or methionine also increased basal activity. PKG1 exists as a parallel homodimer linked by an N-terminal leucine zipper, and we show that the WT chain in WT-RQ heterodimers partly reduces basal activity of the RQ chain. Using hydrogen/deuterium-exchange MS, we found that the RQ substitution causes PKG1β to adopt an active conformation in the absence of cGMP, similar to that of cGMP-bound WT enzyme. We conclude that the RQ substitution in PKG1 increases its basal activity by disrupting the formation of an inactive conformation.

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